The objective of these studies is to identify molecular mechanisms underlying specific stage transitions in multistage carcinogenesis. How transformation of preneoplastic epithelial cells depends on phosphorylation of specific proteins by the phospholipid- and calcium-dependent protein kinase (PK-C) has been the first primary question. Over 16 substrates for PK-C have been found in JB-6 cell lines. Most of them reversibly associate with the particulate fraction depending on the availability of divalent cations. All are more readily phosphorylated under cation conditions favoring phosphorylation of the basic histone, H1 (5.0mM Ca++, 7.5 mM Mg++), but most specifically in a phospholipid dependent manner under conditions favoring the more neutral histones, H2-H4 (5.0mM Ca++, 7.5 mM Mg++). One heat shock protein, pp80, is stimulated by TPA in untransformed but not in transformed derivatives of JB-6 cells. Hyperthermia induces a full heat shock response and blocks TPA promotion. The relationship between this defect in heat shock protein regulation and the synergistic anti-tumor effects of interferon and hyperthermia is being pursued. The generation of reactive oxygen, especially the superoxide anion, has been found to be a required event in TPA-induced promotion in JB-6 cells. Secondary free radicals implicated in this activity are OH-hydroxyl radicals and lipid peroxides but not hydrogen peroxide.